Preparation method of integrated gradual change type bionic gradient structure rotator cuff patch

Abstract

The invention belongs to the technical field of medical materials, and relates to a preparation method of an integrated gradual change type bionic gradient structure rotator cuff patch. The integrated gradual change type bionic gradient structure rotator cuff patch is prepared through the electric auxiliary solution blowing spinning technology and the heat treatment nanoimprint technology, the patch is of a composite layer structure, each transition layer and the adjacent micro-nano fiber membrane layer form a continuous fracture-free structure, the internal structure and component change are in uniform transition, discrete layering is avoided, and the patch has good application prospects. The whole patch is of a continuous gradual change type biological bionic gradient structure, histological gradient characteristics of a tendon-bone transition area are successfully simulated, and rapid healing of a tendon-bone interface can be effectively promoted.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical materials, and relates to a preparation method of an integrated gradient bionic gradient structure rotator cuff patch. Background Art

[0002] Rotator cuff tear is a common tendon injury in orthopedics, with a prevalence rate of 15%-51%, commonly seen in middle-aged and elderly people and those engaged in repetitive upper limb activities. According to the depth of the tear, it is divided into partial tear (not penetrating the full thickness, resulting in a decrease in tendon thickness and strength) and full-thickness tear (complete rupture of the tendon), and the tendon-bone interface is the main injury area.

[0003] The tendon-bone interface is the connection part between the tendon and the bone, with a thickness of about 1 mm, and is composed of four closely connected gradient regions. From the tendon to the bone, they are: Region I is the tendon region, mainly composed of highly oriented fibroblast-like tenocytes, and the extracellular matrix is mainly type I collagen; Region II (unmineralized fibrocartilage) and Region III (mineralized fibrocartilage) together constitute the fibrocartilage zone, which serves as the mineralization front and the junction between hard and soft tissues. The cells in this region are fibrocartilage cells, and their morphology becomes more hypertrophic as the degree of mineralization increases. The extracellular matrix is rich in type II collagen, a large amount of proteoglycans and leucine-rich proteins; Region IV is the bone region, composed of osteoblasts, osteocytes and osteoclasts. On the basis of being rich in type I collagen, the extracellular matrix also significantly contains calcium salt deposition. It is precisely due to the existence of this gradient structure that the tendon-bone interface can act as an "energy dissipator" at the connection part between the tendon and the bone to avoid stress concentration. At the same time, it is also due to the complexity of this gradient structure that the repair of this interface is very difficult.

[0004] Currently, the repair of rotator cuff tear mainly focuses on the physical connection reinforcement between the tendon and the bone, and insufficient attention is paid to the reconstruction of the gradient structure of the tendon-bone interface. Clinical repair strategies include conservative treatment and surgical treatment: Conservative treatment relieves symptoms through immobilization, drugs and physical therapy, but there are problems such as long treatment courses, easy treatment delay, and inability to cure; Surgical treatment includes two techniques: arthroscopic suture repair and patch augmentation implantation surgery. Although suture repair is simple to operate, the retear rate is high and it is only applicable to medium and small tears; Although patch augmentation implantation can partially replace the function of the rotator cuff, due to the single structure of the existing patch, it cannot guide gradient reconstruction, resulting in the formation of scar tissue with poor mechanical properties at the repair site, lacking the stress buffering function of the natural interface, and being prone to micro-injury and even fracture in the long term. Therefore, realizing the reconstruction of the gradient structure of the tendon-bone interface is the key to solving postoperative recurrence and functional recovery.

[0005] In the research of artificial bionic patch implants, the nanofiber membrane prepared by electrospinning technology has shown significant application potential in the simulation and construction of the tendon-bone interface gradient due to its highly controllable, high porosity, high specific surface area, and extracellular matrix-like structure characteristics. However, several problems have also emerged in its practical application. For example, during the preparation process, the difficulty in stacking the thickness of the nanofiber membrane directly affects the quality and performance of its one-piece molding. Therefore, it is often necessary to stack multiple layers of nanofiber membranes to obtain a gradient structure with sufficient thickness. However, the limited gradient simulation degree and poor integrity caused by the discontinuous rather than gradual transition between multiple gradient structures may lead to a reduction in its in vivo functionality, stability, and durability. In addition, low production efficiency is also a defect that cannot be ignored, which greatly limits its application in large-scale production and clinical practice. In addition to electrospinning technology, methods such as air jet spinning technology, 3D printing technology, and combination with traditional textile technology have, to a certain extent, solved the problem of difficult thickness stacking faced by electrospinning technology and achieved certain results in improving mechanical properties. However, these technologies still have limitations. For example, the technical precision is relatively low, and it is difficult to achieve precise construction of microstructures, which is a major defect for the requirement of highly precise simulation of the tendon-bone interface gradient.

[0006] Therefore, for artificial bionic patches, achieving effective and precise simulation and construction of the tendon-bone interface gradient is still a difficult problem that urgently needs to be solved at present. Summary of the Invention

[0007] The purpose of the present invention is to solve the problems existing in the prior art and provide a preparation method for an integrated and gradually changing bionic gradient structure rotator cuff patch.

[0008] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0009] A preparation method for an integrated and gradually changing bionic gradient structure rotator cuff patch. After sequentially forming the first micro-nanofiber membrane layer, the first transition layer, the second micro-nanofiber membrane layer, the second transition layer, the third micro-nanofiber membrane layer, the third transition layer, and the fourth micro-nanofiber membrane layer on a receiving substrate by electro-assisted solution blowing spinning technology, a bone-promoting material protrusion is formed on the fourth micro-nanofiber membrane layer by heat treatment nanoimprinting technology.

[0010] When preparing the i-th micro-nanofiber membrane layer, the corresponding i-th spinning solution is injected at a constant injection speed v i for injection, where i = 1, 2, 3; when preparing the (i + 1)-th micro-nanofiber membrane layer, the corresponding (i + 1)-th spinning solution is injected at a constant injection speed v i+1 for injection; the process of preparing the i-th transition layer is: simultaneously injecting the i-th spinning solution and the (i + 1)-th spinning solution, and the injection speed of the i-th spinning solution changes from v iIt decreases uniformly to 0, and the injection speed of the (i + 1)-th spinning solution increases uniformly from 0 to v. i+1 , and the sum of the injection speeds of the i-th spinning solution and the (i + 1)-th spinning solution at each moment remains unchanged; if the process of preparing the i-th transition layer is as follows: injecting the i-th spinning solution and the (i + 1)-th spinning solution simultaneously, the injection speed of the i-th spinning solution decreases gradientually from v i to 0 (that is, first decreasing to a value and maintaining for a certain time, then continuing to decrease to a value and maintaining for a certain time...), and the injection speed of the (i + 1)-th spinning solution increases gradientually from 0 to v i+1 , and the sum of the injection speeds of the i-th spinning solution and the (i + 1)-th spinning solution at each moment remains unchanged, then it is impossible to ensure that each transition layer and the two adjacent micro-nano fiber membrane layers form a continuous and unbroken structure, with uniform transition of the internal structure and composition, and no discrete stratification, because the transition layer prepared in this way is formed by superimposing multiple sub-layers, and there is still a discontinuous transition between layers in essence;

[0011] The first micro-nano fiber membrane layer is used to guide the directional arrangement of tendon fibroblasts and promote tendon differentiation and regeneration; the second micro-nano fiber membrane layer is used to support and induce the differentiation and regeneration of unmineralized fibrocartilage; the third micro-nano fiber membrane layer is used to induce the differentiation and regeneration of mineralized fibrocartilage; the fourth micro-nano fiber membrane layer is used to promote bone regeneration; the osteogenic material protrusions are cylindrical, with a diameter of 100 - 2500 nm and a height of 5 - 2500 nm. All the osteogenic material protrusions are arranged in a dot matrix, and the distance between two adjacent osteogenic material protrusions is 100 - 2000 nm. The osteogenic material protrusions are used to further enhance the mineralization near the bone side of the patch, simulate the microenvironment of natural bone tissue, enhance cell adhesion and proliferation, and promote bone regeneration and bone bonding; the biological properties of the first micro-nano fiber membrane layer are designed to simulate the tendon tissue in the rotator cuff tendon-bone interface, and its fiber structure and material composition are designed to provide biomechanical properties similar to those of tendons; the biological properties of the second micro-nano fiber membrane layer are designed to simulate the unmineralized fibrocartilage tissue in the rotator cuff tendon-bone interface, and its structural characteristics and osteogenic material concentration are designed to simulate the biological activity of unmineralized cartilage; the biological properties of the third micro-nano fiber membrane layer are designed to simulate the mineralized fibrocartilage tissue in the rotator cuff tendon-bone interface, and its fiber arrangement and material composition are designed to reproduce the hardness of mineralized cartilage and the biological micro-nano fiber membrane; the biological properties of the fourth micro-nano fiber membrane layer and the osteogenic material protrusions are designed to simulate the bone tissue in the rotator cuff tendon-bone interface, and its structure and osteogenic material concentration are designed to promote the growth and integration of bone tissue;

[0012] Each transition layer and the two adjacent micro-nano fiber membrane layers form a continuous and unbroken structure, with uniform transition of the internal structure and composition, and no discrete stratification.

[0013] The integrated gradient bionic structure rotator cuff patch is a three-dimensional structure, and the overall structure is one or more of a cube, a cuboid, a cylinder, and a polyhedron;

[0014] Compared with the conventional patch with a single uniform structure, the multi-layer composite structure of the patch of the present invention exhibits more delicate biomechanical compatibility and biodegradability in the process of promoting tendon-bone healing and tissue regeneration, and significantly reduces the incidence of secondary tears.

[0015] The electro-assisted solution blow spinning technology combines the advantages of air jet spinning technology and electrospinning technology, and has the advantages of simple process, high efficiency and controllability, wide applicability, etc. It can accurately control micro-nano scale fibers while greatly improving production efficiency, and can achieve material stacking with a thickness of millimeters.

[0016] The integrated gradient bionic rotator cuff patch is prepared by electro-assisted solution blow spinning technology, realizing integrated molding. The whole patch presents a continuous gradient bionic structure: in the horizontal dimension, each micro-nano fiber membrane layer maintains the uniformity of fiber morphological parameters and chemical composition; in the vertical dimension, it shows the variation of structural parameters such as fiber diameter, orientation degree, porosity, etc. and the concentration gradient change of osteoinductive materials, successfully simulating the histological gradient characteristics of the tendon-bone transition region, and finally realizing the biological functional simulation of the rotator cuff tendon-bone interface.

[0017] As a preferred technical solution:

[0018] For the preparation method of an integrated gradient bionic structure rotator cuff patch as described above, the average pore size of the first micro-nano fiber membrane layer is 10-50 μm to allow and facilitate the infiltration of tendon fibroblasts, the porosity is 70-85% to ensure the uniform distribution of cells and nutrients, the orientation degree of fiber arrangement >80% to simulate the collagen arrangement of natural tendons, the fiber component is a mixture of natural polymer materials and synthetic polymer materials to simulate the biological characteristics of tendon tissue, and the content of the synthetic polymer material is 10-90 wt%.

[0019] For the preparation method of an integrated gradient bionic structure rotator cuff patch as described above, the average pore size of the second micro-nano fiber membrane layer is 30-80 μm to promote the residence of chondrocytes / stem cells. Specifically, the smaller pore size and limited infiltration space can induce the chondrogenesis of MSCs, resulting in the formation of cartilage tissue rather than bone tissue. The porosity is 80-90%, the orientation degree of fiber arrangement is 50-60% to form an anisotropic structure, and the fiber component is a synthetic polymer material.

[0020] A preparation method of an integrated gradient bionic gradient structure rotator cuff patch as described above. The average pore size of the third micro-nano fiber membrane layer is 100 - 300 μm to facilitate blood vessel invasion and mineralization, the porosity is 85 - 95%, and the orientation degree of fiber arrangement is greater than 20% and less than 30% to form a porous scaffold. The fiber component is a mixture of a synthetic polymer material and an osteoinductive material, and the content of the osteoinductive material is 1 - 2 wt%.

[0021] A preparation method of an integrated gradient bionic gradient structure rotator cuff patch as described above. The average pore size of the fourth micro-nano fiber membrane layer is 200 - 500 μm, meeting the requirements of bone tissue engineering, the porosity > 90%, the fibers are randomly oriented, and the fiber component is a mixture of a natural polymer material, a synthetic polymer material, and an osteoinductive material. The content of the synthetic polymer material is 10 - 90 wt%, and the content of the osteoinductive material is 2 - 5 wt%.

[0022] A preparation method of an integrated gradient bionic gradient structure rotator cuff patch as described above. The natural polymer material is one or more of type I collagen, gelatin, chitosan, silk fibroin, hyaluronic acid, alginate, cellulose, and its derivatives;

[0023] The synthetic polymer material is one or more of polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), poly(p-dioxanone) (PDO), poly(lactic-co-caprolactone) (PLCL), and polyethylene glycol (PEG). When the synthetic polymer material is PCL, it will melt after thermal nanoimprinting treatment to enhance the mechanical properties;

[0024] The osteoinductive material includes but is not limited to osteoactive drugs, osteoactive growth factors, and inorganic osteoinductive materials, such as one or more of nano-hydroxyapatite, struvite nanowires, bioactive glass, and tricalcium phosphate;

[0025] The thickness of each micro-nano fiber membrane layer ranges from 0.1 - 1 mm, and the total thickness of the integrated gradient bionic gradient structure rotator cuff patch is 0.7 - 6 mm.

[0026] A preparation method of an integrated gradient bionic gradient structure rotator cuff patch as described above. The specific steps of forming osteoinductive material protrusions on the fourth micro-nano fiber membrane layer by using the heat treatment nanoimprinting technique are as follows:

[0027] (a) Pour the photosensitive resin onto the nano-pattern master, spread it evenly with a roller until the surface of the nano-pattern master is completely covered, and then cure the photosensitive resin (first perform photocuring treatment on the photosensitive resin with an ultraviolet light source, and then place it in an oven for heating for post-curing treatment). After curing, a resin mold is formed on the nano-pattern master, and the nano-pattern master is separated from the resin mold;

[0028] (b) Deposit osteoinductive material on the surface of the resin mold by electron beam evaporation technology;

[0029] (c) Perform plasma treatment on the resin mold with osteoinductive material deposited on its surface to reduce the nano-pattern width, thereby reducing the interfacial adhesion force between the resin mold and the osteoinductive material. At the same time, perform plasma treatment on the fourth micro-nano fiber membrane layer to promote the formation of hydroxyl groups and increase the surface energy, thereby enhancing the adhesion of the fourth micro-nano fiber membrane layer;

[0030] (d) Press the resin mold with osteoinductive material deposited on its surface onto the fourth micro-nano fiber membrane layer. After the osteoinductive material comes into contact with the fourth micro-nano fiber membrane layer, perform heat treatment. After the heat treatment is completed, remove the resin mold.

[0031] Heat treatment nanoimprinting technology combines the heat treatment process with the mold pattern. First, heat-treat the polymer material to make it soft, and then, under the action of an external pressure, accurately transfer the nano-pattern while maintaining the integrity of the material's main structure. This high-precision micro-nano pattern construction technology can not only enhance the mechanical properties of the material but also precisely regulate the micro-nano structure on the material surface to achieve functional modification.

[0032] A preparation method of an integrated gradient bionic gradient structure rotator cuff patch as described above. The process parameters of electro-assisted solution blow spinning include: positive voltage of 10 - 18 kV, negative voltage of 1 - 3 kV, injection speed of 1 - 10 mL / h, air pressure of 1 - 10 atm, and receiving distance of 10 - 20 cm.

[0033] Beneficial effects:

[0034] (1) The integrated gradient bionic gradient structure rotator cuff patch prepared by the present invention has a composite layer structure, and each transition layer and the adjacent micro-nano fiber membrane layer form a continuous and unbroken structure. The internal structure and composition change uniformly without presenting discrete stratification. The overall patch presents a continuous gradient bionic gradient structure, with gradient-changing mechanical properties and tissue induction properties, and can achieve the adhesion, proliferation, and differentiation of tendon tissue and bone tissue respectively in different functional layers of the patch, successfully simulating the histological gradient characteristics of the tendon-bone transition region and realizing the biological functional simulation of the rotator cuff tendon-bone interface. Compared with conventional patches with a single uniform structure, the integrated gradient bionic gradient structure rotator cuff patch prepared by the present invention can effectively promote the rapid healing of the tendon-bone interface and significantly reduce the incidence of secondary tears.

[0035] (2) The electrically assisted solution blowing spinning technology used in the present invention combines the advantages of air-jet spinning and electrospinning, and has the characteristics of simple process, high efficiency and controllability, and wide applicability. This technology not only has a continuous and stable spinning process and high production capacity, but also can accurately control the morphology and size of micro-nano fibers. At the same time, due to the synergistic effect of air flow force and electric field force, this technology can handle high-viscosity and high-concentration spinning precursor solutions, achieve efficient accumulation of materials with millimeter-level thickness, and significantly improve production efficiency. In addition, this technology is used to prepare an integrated gradient bionic gradient structure shoulder cuff patch, realizing the integrated molding of the patch.

[0036] (3) The heat treatment nanoimprinting technology used in the present invention combines heat treatment process, electron beam evaporation technology, plasma surface modification technology and mold pattern, which can achieve the precise inlay and transfer of the nano-pattern of the bone-promoting material on the surface of the nanoimprint template to the surface of the fourth micro-nano fiber membrane layer of the patch, while maintaining the integrity of the material body structure. This high-precision micro-nano pattern construction technology can not only enhance the mechanical properties of the patch, but also accurately control the micro-nano structure of the material surface, further enhance the mineralization degree of the bone regeneration functional layer, and achieve functional modification to promote bone healing. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is the cross-sectional SEM of the integrated gradual bionic gradient structure rotator cuff patch prepared in Example 1. DETAILED DESCRIPTION

[0038] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0039] The following are the test methods for the relevant performance indicators in each embodiment:

[0040] Pore ​​size and porosity: A mercury intrusion instrument is used to test the pore size and porosity of the patch. A sample of at least 0.1 g is cut into pieces and placed in a low-pressure chamber to evacuate the residual gas. The mercury injection pressure is then gradually increased through the hydraulic system, and the volume increment of mercury infiltrated into the sample under different pressures is simultaneously recorded. The pore size and porosity data are obtained through the test software provided by the computer.

[0041] Orientation degree of fiber arrangement: A scanning electron microscope was used to obtain high-resolution two-dimensional images of the samples, and then Image J software was used to extract the fiber orientation angle and statistically analyze the fiber orientation distribution.

[0042] Peeling strength: To characterize that each transition layer of the patch and the two adjacent micro-nano fiber membrane layers form a continuous and unbroken structure, the bonding strength between the layers of the sample to be tested is measured at room temperature according to a variant standard of the ASTM D1876-72(1983) T-peel test using a multi-functional tensile tester; the patch is cut into a rectangle of 20 mm × 10 mm, and the initial gauge length of the instrument is adjusted according to the actual thickness of the patch; the upper and lower surfaces of one end of the patch are respectively fixed to the lower surface of the upper sensor and the upper surface of the lower rigid material platform using strong glue, a 100 N sensor is used, and the peeling test is carried out at a tensile speed of 10 mm / min and a pre-tension of 0.1 N.

[0043] Energy dispersive spectroscopy analysis: Using a field emission transmission electron microscope, energy dispersive spectroscopy analysis (EDS) is carried out on each layer of the sample to be tested under the condition of an acceleration voltage of 200 kV.

[0044] The source information of the relevant substances in the following examples is shown in Table 1:

[0045] Table 1

[0046]

[0047] Example 1

[0048] A preparation method of an integrated gradient bionic gradient structure rotator cuff patch, the specific steps are as follows:

[0049] (1) Preparation of materials;

[0050] The first spinning solution: The solute is a mixture of type I collagen and PDLLA, the content of PDLLA in the solute is 10 wt%, the solvent is a mixture of hexafluoroisopropanol and acetic acid with a volume ratio of 5:5, and the concentration is 100 mg / mL;

[0051] The second spinning solution: The solute is PDLLA, the solvent is hexafluoroisopropanol, and the concentration is 110 mg / mL;

[0052] The third spinning solution: The solute is a mixture of PDLLA and osteoinductive material a, the content of osteoinductive material a in the solute is 1 wt%, the solvent is hexafluoroisopropanol, and the concentration is 120 mg / mL;

[0053] The fourth spinning solution: The solute is a mixture of type I collagen, PDLLA and osteoinductive material b, the content of PDLLA in the solute is 10 wt%, the content of osteoinductive material b in the solute is 2 wt%, the solvent is a mixture of hexafluoroisopropanol and acetic acid with a volume ratio of 5:5, and the concentration is 130 mg / mL;

[0054] Osteoinductive material a, osteoinductive material b and osteoinductive material c: All are struvite nanowires;

[0055] (2) The electro-assisted solution blow spinning technology is adopted to sequentially form a first micro-nano fiber membrane layer, a first transition layer, a second micro-nano fiber membrane layer, a second transition layer, a third micro-nano fiber membrane layer, a third transition layer, and a fourth micro-nano fiber membrane layer on a receiving substrate;

[0056] When preparing the first micro-nano fiber membrane layer, the first spinning solution is injected at a constant injection speed v 1 for injection; the process parameters include: the positive voltage is 10 kV, the negative voltage is 1 kV, v 1 is 1 mL / h, the air pressure is 1 atm, and the receiving distance is 10 cm; the thickness of the first micro-nano fiber membrane layer is 0.1 mm, the average pore diameter is 10 μm, the porosity is 70%, and the fiber alignment degree is 80%; the first micro-nano fiber membrane layer is used to guide the directional arrangement of tendon fibroblasts and promote tendon differentiation and regeneration;

[0057] When preparing the first transition layer, the first spinning solution and the second spinning solution are injected simultaneously. The injection speed of the first spinning solution decreases uniformly from v 1 to 0, and the injection speed of the second spinning solution increases uniformly from 0 to v 2 , and the sum of the injection speeds of the first spinning solution and the second spinning solution at each moment remains unchanged; the process parameters include: the positive voltage is 10 kV, the negative voltage is 1 kV, v 2 is 1 mL / h, the injection time is 1 h, the air pressure is 1 atm, and the receiving distance is 10 cm;

[0058] When preparing the second micro-nano fiber membrane layer, the second spinning solution is injected at a constant injection speed v 2 for injection; the process parameters include: the positive voltage is 10 kV, the negative voltage is 1 kV, the air pressure is 1 atm, and the receiving distance is 10 cm; the thickness of the second micro-nano fiber membrane layer is 0.1 mm, the average pore diameter is 30 μm, the porosity is 80%, and the fiber alignment degree is 53%; the second micro-nano fiber membrane layer is used to support and induce the differentiation and regeneration of unmineralized fibrous cartilage;

[0059] When preparing the second transition layer, the second spinning solution and the third spinning solution are injected simultaneously. The injection speed of the second spinning solution decreases uniformly from v 2 to 0, and the injection speed of the second spinning solution increases uniformly from 0 to v 3 , and the sum of the injection speeds of the second spinning solution and the third spinning solution at each moment remains unchanged; the process parameters include: the positive voltage is 10 kV, the negative voltage is 1 kV, v 3 is 1 mL / h, the injection time is 1 h, the air pressure is 1 atm, and the receiving distance is 10 cm;

[0060] When preparing the third micro-nano fiber membrane layer, the third spinning solution is injected at a constant pushing speed v. 3 The injection is carried out; the process parameters include: the positive voltage is 10 kV, the negative voltage is 1 kV, the air pressure is 1 atm, and the receiving distance is 10 cm; the thickness of the third micro-nano fiber membrane layer is 0.1 mm, the average pore diameter is 100 μm, the porosity is 88%, and the fiber alignment degree is 23%; the third micro-nano fiber membrane layer is used to induce mineralized fiber cartilage differentiation and regeneration.

[0061] When preparing the third transition layer, the third spinning solution and the fourth spinning solution are injected simultaneously. The pushing speed of the third spinning solution decreases uniformly from v 3 to 0, and the pushing speed of the fourth spinning solution increases uniformly from 0 to v. 4 At each moment, the sum of the pushing speeds of the third spinning solution and the fourth spinning solution remains unchanged; the process parameters include: the positive voltage is 10 kV, the negative voltage is 1 kV, v 4 is 1 mL / h, the injection time is 1 h, the air pressure is 1 atm, and the receiving distance is 10 cm.

[0062] When preparing the fourth micro-nano fiber membrane layer, the fourth spinning solution is injected at a constant pushing speed v. 4 The injection is carried out; the process parameters include: the positive voltage is 10 kV, the negative voltage is 1 kV, the air pressure is 1 atm, and the receiving distance is 10 cm; the thickness of the fourth micro-nano fiber membrane layer is 0.1 mm, the average pore diameter is 200 μm, the porosity is 90%, and the fibers are completely randomly oriented; the fourth micro-nano fiber membrane layer is used to promote bone regeneration.

[0063] (3) A bone-promoting material c bulge is formed on the fourth micro-nano fiber membrane layer by using a heat treatment nanoimprinting technique.

[0064] (3.1) Pour the photosensitive resin onto the nano-pattern master, spread it evenly with a roller until it completely covers the surface of the nano-pattern master, and then cure the photosensitive resin. After curing, a resin mold is formed on the nano-pattern master, and the nano-pattern master and the resin mold are separated.

[0065] (3.2) Deposit the bone-promoting material c on the surface of the resin mold by using an electron beam evaporation technique.

[0066] (3.3) Perform plasma treatment on the resin mold with the bone-promoting material c deposited on its surface, and simultaneously perform plasma treatment on the fourth micro-nano fiber membrane layer.

[0067] (3.4) Press the resin mold with the bone-promoting material c deposited on its surface onto the fourth micro-nano fiber membrane layer. After the bone-promoting material c contacts the fourth micro-nano fiber membrane layer, perform heat treatment. After the heat treatment is completed, remove the resin mold to obtain an integrated gradient bionic gradient structure rotator cuff patch.

[0068] The total thickness of the finally obtained integrated gradient bionic gradient structure rotator cuff patch is 0.7 mm; the osteogenic material c protrusions on the fourth micro-nano fiber membrane layer are cylindrical, with a diameter of 100 nm and a height of 5 nm. All the osteogenic material c protrusions are arranged in a dot matrix, and the distance between adjacent two osteogenic material c protrusions is 100 nm; each transition layer and the two adjacent micro-nano fiber membrane layers form a continuous and unbroken structure, and the internal structure and composition change uniformly without showing discrete stratification (the cross-sectional SEM of the integrated gradient bionic gradient structure rotator cuff patch is as Figure 1 shown);

[0069] The peeling strength of the integrated gradient bionic gradient structure rotator cuff patch is 48.3 mN / mm; the energy dispersive spectroscopy analysis of each layer of the integrated gradient bionic gradient structure rotator cuff patch is shown in Table 2:

[0070] Table 2

[0071]

[0072] As can be seen from Table 2, the first micro-nano fiber membrane layer is mainly dominated by type I collagen, with a high N content, and PDLLA slightly increases the C ratio; in the first transition layer, the PDLLA content increases, C increases and N decreases; the solute in the second micro-nano fiber membrane layer only contains PDLLA, and the C and O ratios are close to the theoretical values; in the second transition layer, Mg, P, and N signals are introduced due to the introduction of trace amounts of struvite nanowires, and C and O are still mainly PDLLA; in the third micro-nano fiber membrane layer, the content of struvite nanowires (osteogenic material a) increases, and the Mg and P signals are significantly enhanced, while the C ratio decreases; in the third transition layer, the increase in type I collagen enhances N, and the increase in struvite nanowires (osteogenic material b) leads to an increase in Mg and P; in the fourth micro-nano fiber membrane layer, the proportion of type I collagen is the highest, N increases significantly, and the struvite nanowires (osteogenic material c) continuously provide Mg and P signals.

[0073] Example 2

[0074] A preparation method of an integrated gradient bionic gradient structure rotator cuff patch, the specific steps are as follows:

[0075] (1) Preparation of materials;

[0076] The first spinning solution: the solute is a mixture of gelatin and PLGA, the content of PLGA in the solute is 30 wt%, the solvent is a mixture of hexafluoroisopropanol and acetic acid with a volume ratio of 7:3, and the concentration is 125 mg / mL;

[0077] The second spinning solution: the solute is PLGA, the solvent is hexafluoroisopropanol, and the concentration is 135 mg / mL; the third spinning solution: the solute is a mixture of PLGA and osteoinductive material a, the content of osteoinductive material a in the solute is 1 wt%, the solvent is hexafluoroisopropanol, and the concentration is 145 mg / mL;

[0078] The fourth spinning solution: the solute is a mixture of gelatin, PLGA and osteoinductive material b, the content of PLGA in the solute is 30 wt%, the content of osteoinductive material b in the solute is 2 wt%, and the solvent is a mixture of hexafluoroisopropanol and acetic acid with a volume ratio of 7:3;

[0079] of the mixture, and the concentration is 155 mg / mL;

[0080] Osteoinductive material a, osteoinductive material b, osteoinductive material c: all are nano-hydroxyapatite;

[0081] (2)Using the electro-assisted solution blow spinning technology, the first micro-nano fiber membrane layer, the first transition layer, the second micro-nano fiber membrane layer, the second transition layer, the third micro-nano fiber membrane layer, the third transition layer, and the fourth micro-nano fiber membrane layer are successively formed on the receiving substrate;

[0082] When preparing the first micro-nano fiber membrane layer, the first spinning solution is pushed at a constant injection speed v 1 for injection; the process parameters include: the positive voltage is 12 kV, the negative voltage is 1 kV, v 1 is 3 mL / h, the air pressure is 3 atm, and the receiving distance is 13 cm; the thickness of the first micro-nano fiber membrane layer is 0.3 mm, the average pore size is 23 μm, the porosity is 75%, and the orientation degree of fiber arrangement is 83%; the first micro-nano fiber membrane layer is used to guide the directional arrangement of tendon fibroblasts and promote tendon differentiation and regeneration;

[0083] When preparing the first transition layer, the first spinning solution and the second spinning solution are simultaneously pushed. The injection speed of the first spinning solution is uniformly reduced from v 1 to 0, and the injection speed of the second spinning solution is uniformly increased from 0 to v 2 , and the sum of the injection speeds of the first spinning solution and the second spinning solution at each moment remains unchanged; the process parameters include: the positive voltage is 12 kV, the negative voltage is 1 kV, v 2 is 3 mL / h, the injection time is 1 h, the air pressure is 3 atm, and the receiving distance is 13 cm;

[0084] When preparing the second micro-nano fiber membrane layer, the second spinning solution is pushed at a constant injection speed v 2Perform bolus injection; the process parameters include: positive voltage of 12 kV, negative voltage of 1 kV, air pressure of 3 atm, and receiving distance of 13 cm; the thickness of the second micro-nano fiber membrane layer is 0.3 mm, the average pore size is 45 μm, the porosity is 82%, and the fiber alignment degree is 60%; the second micro-nano fiber membrane layer is used to support and induce the differentiation and regeneration of unmineralized fibrous cartilage;

[0085] When preparing the second transition layer, simultaneously inject the second spinning solution and the third spinning solution. The injection speed of the second spinning solution decreases uniformly from v 2 to 0 at a uniform speed, and the injection speed of the second spinning solution increases uniformly from 0 to v 3 , and the sum of the injection speeds of the second spinning solution and the third spinning solution remains unchanged at each moment; the process parameters include: positive voltage of 12 kV, negative voltage of 1 kV, v 3 is 3 mL / h, the injection time is 1 h, the air pressure is 3 atm, and the receiving distance is 13 cm;

[0086] When preparing the third micro-nano fiber membrane layer, inject the third spinning solution at a constant injection speed v 3 ; the process parameters include: positive voltage of 12 kV, negative voltage of 1 kV, air pressure of 3 atm, and receiving distance of 13 cm; the thickness of the third micro-nano fiber membrane layer is 0.3 mm, the average pore size is 135 μm, the porosity is 85%, and the fiber alignment degree is 30%; the third micro-nano fiber membrane layer is used to induce the differentiation and regeneration of mineralized fibrous cartilage;

[0087] When preparing the third transition layer, simultaneously inject the third spinning solution and the fourth spinning solution. The injection speed of the third spinning solution decreases uniformly from v 3 to 0 at a uniform speed, and the injection speed of the second spinning solution increases uniformly from 0 to v 4 , and the sum of the injection speeds of the third spinning solution and the fourth spinning solution remains unchanged at each moment; the process parameters include: positive voltage of 12 kV, negative voltage of 1 kV, v 4 is 3 mL / h, the injection time is 1 h, the air pressure is 3 atm, and the receiving distance is 13 cm;

[0088] When preparing the fourth micro-nano fiber membrane layer, inject the fourth spinning solution at a constant injection speed v 4 ; the process parameters include: positive voltage of 12 kV, negative voltage of 1 kV, air pressure of 3 atm, and receiving distance of 13 cm; the thickness of the fourth micro-nano fiber membrane layer is 0.3 mm, the average pore size is 271 μm, the porosity is 91%, and the fibers are completely randomly oriented; the fourth micro-nano fiber membrane layer is used to promote bone regeneration;

[0089] (3) The osteogenic material c protrusions are formed on the fourth micro-nano fiber membrane layer by using the heat treatment nanoimprinting technique;

[0090] (3.1) Pour the photosensitive resin onto the nano-patterned master, spread it evenly with a roller until the surface of the nano-patterned master is completely covered, then cure the photosensitive resin. After the curing treatment, a resin mold is formed on the nano-patterned master, and the nano-patterned master is separated from the resin mold;

[0091] (3.2) Deposit the osteogenic material c on the surface of the resin mold by using the electron beam evaporation technique;

[0092] (3.3) Perform plasma treatment on the resin mold with the osteogenic material c deposited on its surface, and at the same time perform plasma treatment on the fourth micro-nano fiber membrane layer;

[0093] (3.4) Press the resin mold with the osteogenic material c deposited on its surface onto the fourth micro-nano fiber membrane layer. After the osteogenic material c comes into contact with the fourth micro-nano fiber membrane layer, perform heat treatment. After the heat treatment is completed, remove the resin mold to obtain the integrated gradient bionic structure rotator cuff patch.

[0094] The total thickness of the finally obtained integrated gradient bionic structure rotator cuff patch is 2.1 mm; the osteogenic material c protrusions on the fourth micro-nano fiber membrane layer are cylindrical, with a diameter of 500 nm and a height of 100 nm. All the osteogenic material c protrusions are arranged in a dot matrix, and the distance between adjacent two osteogenic material c protrusions is 500 nm; each transition layer and the two adjacent micro-nano fiber membrane layers form a continuous and unbroken structure, and the internal structure and composition change evenly without presenting discrete stratification;

[0095] The peel strength of the integrated gradient bionic structure rotator cuff patch is 51.4 mN / mm; the energy dispersive spectroscopy analysis of each layer of the integrated gradient bionic structure rotator cuff patch is shown in Table 3:

[0096] Table 3

[0097]

[0098] As can be seen from Table 3, in the first micro-nano fiber membrane layer, gelatin, as the main component, contributed N element, and the presence of PLGA increased the proportion of C element; in the first transition layer, with the increase of PLGA content, the proportions of C and O elements increased, and the N content decreased due to the decrease in the proportion of gelatin; in the second micro-nano fiber membrane layer, the solute only contained PLGA, and the proportions of C and O elements were close to 1:1 (resulting from the alternating arrangement of LA and GA units); in the second transition layer, due to the introduction of a small amount of nano-hydroxyapatite (osteogenic material a), Ca and P signals appeared, but the C and O elements were still mainly contributed by PLGA; in the third micro-nano fiber membrane layer, the proportion of nano-hydroxyapatite (osteogenic material a) further increased, the Ca and P signals were significantly enhanced, and at the same time the proportion of C element decreased; in the third transition layer, the recovery of gelatin component caused the N element signal to rise, and the continuous addition of nano-hydroxyapatite (osteogenic material b) further increased the Ca and P contents; in the fourth micro-nano fiber membrane layer, the proportion of gelatin reached the highest, the N content increased significantly, and nano-hydroxyapatite (osteogenic material c) continuously provided Ca and P signals.

[0099] Example 3

[0100] A preparation method of an integrated gradient bionic gradient structure rotator cuff patch, the specific steps are as follows:

[0101] (1)Preparation of materials;

[0102] The first spinning solution: the solute is a mixture of chitosan and PCL, the content of PCL in the solute is 50 wt%, the solvent is a mixture of hexafluoroisopropanol and acetic acid aqueous solution with a volume ratio of 7:3 (the volume fraction of acetic acid is 80%), and the concentration is 150 mg / mL;

[0103] The second spinning solution: the solute is PCL, the solvent is hexafluoroisopropanol, and the concentration is 160 mg / mL;

[0104] The third spinning solution: the solute is a mixture of PCL and osteogenic material a, the content of osteogenic material a in the solute is 2 wt%, the solvent is hexafluoroisopropanol, and the concentration is 170 mg / mL;

[0105] The fourth spinning solution: the solute is a mixture of chitosan, PCL and osteogenic material b, the content of PCL in the solute is 50 wt%, the content of osteogenic material b in the solute is 3 wt%, the solvent is a mixture of hexafluoroisopropanol and acetic acid aqueous solution with a volume ratio of 7:3 (the volume fraction of acetic acid is 80%), and the concentration is 180 mg / mL;

[0106] Osteogenic material a, osteogenic material b, osteogenic material c: all are nano-hydroxyapatite;

[0107] (2)The electro-assisted solution blowing spinning technology is adopted to sequentially form the first micro-nano fiber membrane layer, the first transition layer, the second micro-nano fiber membrane layer, the second transition layer, the third micro-nano fiber membrane layer, the third transition layer, and the fourth micro-nano fiber membrane layer on the receiving substrate;

[0108] When preparing the first micro-nano fiber membrane layer, the first spinning solution is pushed at a constant injection speed v 1 for injection; the process parameters include: the positive voltage is 14 kV, the negative voltage is 2 kV, v 1 is 5 mL / h, the air pressure is 5 atm, and the receiving distance is 15 cm; the thickness of the first micro-nano fiber membrane layer is 0.5 mm, the average pore diameter is 31 μm, the porosity is 80%, and the fiber alignment degree is 82%; the first micro-nano fiber membrane layer is used to guide the directional arrangement of tendon fibroblasts and promote tendon differentiation and regeneration;

[0109] When preparing the first transition layer, the first spinning solution and the second spinning solution are simultaneously pushed. The injection speed of the first spinning solution decreases uniformly from v 1 to 0, and the injection speed of the second spinning solution increases uniformly from 0 to v 2 , and the sum of the injection speeds of the first spinning solution and the second spinning solution at each moment remains unchanged; the process parameters include: the positive voltage is 14 kV, the negative voltage is 2 kV, v 2 is 5 mL / h, the injection time is 1 h, the air pressure is 5 atm, and the receiving distance is 15 cm;

[0110] When preparing the second micro-nano fiber membrane layer, the second spinning solution is pushed at a constant injection speed v 2 for injection; the process parameters include: the positive voltage is 14 kV, the negative voltage is 2 kV, the air pressure is 5 atm, and the receiving distance is 15 cm; the thickness of the second micro-nano fiber membrane layer is 0.5 mm, the average pore diameter is 58 μm, the porosity is 85%, and the fiber alignment degree is 56%; the second micro-nano fiber membrane layer is used to support and induce the differentiation and regeneration of unmineralized fibrous cartilage;

[0111] When preparing the second transition layer, the second spinning solution and the third spinning solution are simultaneously pushed. The injection speed of the second spinning solution decreases uniformly from v 2 to 0, and the injection speed of the second spinning solution increases uniformly from 0 to v 3 , and the sum of the injection speeds of the second spinning solution and the third spinning solution at each moment remains unchanged; the process parameters include: the positive voltage is 14 kV, the negative voltage is 2 kV, v 3 is 5 mL / h, the injection time is 1 h, the air pressure is 5 atm, and the receiving distance is 15 cm;

[0112] When preparing the third micro-nano fiber membrane layer, the third spinning solution is pushed at a constant injection speed v3 Perform a bolus injection; the process parameters include: a positive voltage of 14 kV, a negative voltage of 2 kV, an air pressure of 5 atm, and a receiving distance of 15 cm; the thickness of the third micro-nano fiber membrane layer is 0.5 mm, the average pore size is 186 μm, the porosity is 95%, and the fiber alignment degree is 23%; the third micro-nano fiber membrane layer is used to induce mineralized fiber cartilage differentiation and regeneration;

[0113] When preparing the third transition layer, simultaneously inject the third spinning solution and the fourth spinning solution. The injection speed of the third spinning solution decreases uniformly from v 3 to 0, and the injection speed of the second spinning solution increases uniformly from 0 to v 4 , and the sum of the injection speeds of the third spinning solution and the fourth spinning solution remains unchanged at each moment; the process parameters include: a positive voltage of 14 kV, a negative voltage of 2 kV, v 4 is 5 mL / h, the injection time is 1 h, the air pressure is 5 atm, and the receiving distance is 15 cm;

[0114] When preparing the fourth micro-nano fiber membrane layer, inject the fourth spinning solution at a constant injection speed v 4 ; the process parameters include: a positive voltage of 14 kV, a negative voltage of 2 kV, an air pressure of 5 atm, and a receiving distance of 15 cm; the thickness of the fourth micro-nano fiber membrane layer is 0.5 mm, the average pore size is 359 μm, the porosity is 92%, and the fibers are completely randomly oriented; the fourth micro-nano fiber membrane layer is used to promote bone regeneration;

[0115] (3) Use the heat treatment nanoimprinting technology to form osteogenic material c protrusions on the fourth micro-nano fiber membrane layer;

[0116] (3.1) Pour the photosensitive resin onto the nano-patterned master, spread it evenly with a roller until the surface of the nano-patterned master is completely covered, then cure the photosensitive resin. After curing, a resin mold is formed on the nano-patterned master, and the nano-patterned master and the resin mold are separated;

[0117] (3.2) Use the electron beam evaporation technology to deposit osteogenic material c on the surface of the resin mold;

[0118] (3.3) Perform plasma treatment on the resin mold with osteogenic material c deposited on its surface, and simultaneously perform plasma treatment on the fourth micro-nano fiber membrane layer;

[0119] (3.4) Press the resin mold with osteogenic material c deposited on its surface onto the fourth micro-nano fiber membrane layer. After osteogenic material c comes into contact with the fourth micro-nano fiber membrane layer, perform heat treatment. After the heat treatment is completed, remove the resin mold to obtain an integrated gradient bionic structure rotator cuff patch.

[0120] The total thickness of the finally obtained integrated gradient bionic structure rotator cuff patch is 3.5 mm; the osteogenic material c protrusions on the fourth micro-nano fiber membrane layer are cylindrical, with a diameter of 800 nm and a height of 200 nm. All the osteogenic material c protrusions are arranged in a dot matrix, and the distance between adjacent two osteogenic material c protrusions is 800 nm; each transition layer and the two adjacent micro-nano fiber membrane layers form a continuous and unbroken structure, and the internal structure and composition change evenly without discrete stratification;

[0121] The peeling strength of the integrated gradient bionic structure rotator cuff patch is 61.7 mN / mm; the energy dispersive spectroscopy analysis of each layer of the integrated gradient bionic structure rotator cuff patch is shown in Table 4:

[0122] Table 4

[0123]

[0124] It can be seen from Table 4 that in the first micro-nano fiber membrane layer, chitosan provides N element as one of the main components, and the presence of PCL significantly increases the proportion of C element; in the first transition layer, as the content of PCL increases, the proportion of C element further rises, and the N content decreases due to the decrease in the proportion of chitosan; in the second micro-nano fiber membrane layer, since the solute only contains PCL, C element dominates, and the ratio of C to O elements is close to the theoretical value (about 69:31); in the second transition layer, due to the introduction of a small amount of nano-hydroxyapatite (osteogenic material a), Ca and P signals begin to appear, but C and O elements still mainly come from PCL; in the third micro-nano fiber membrane layer, as the content of nano-hydroxyapatite (osteogenic material a) increases, the Ca and P signals are significantly enhanced; in the third transition layer, the recovery of chitosan component makes the trace N signal rise, and at the same time, the proportion of nano-hydroxyapatite (osteogenic material b) increases, further enhancing the Ca and P signals; in the fourth micro-nano fiber membrane layer, the combined action of chitosan and nano-hydroxyapatite (osteogenic material c) makes the N element and the contents of Ca and P increase simultaneously.

[0125] Example 4

[0126] A preparation method of an integrated gradient bionic structure rotator cuff patch, the specific steps are as follows:

[0127] The first spinning solution: the solute is a mixture of silk fibroin and PDO, the content of PDO in the solute is 70 wt%, the solvent is a mixture of hexafluoroisopropanol and formic acid with a volume ratio of 8:2, and the concentration is 175 mg / mL;

[0128] The second spinning solution: the solute is PDO, the solvent is hexafluoroisopropanol, and the concentration is 185 mg / mL;

[0129] The third spinning solution: The solute is a mixture of PDO and osteoinductive material a, the content of osteoinductive material a in the solute is 2 wt%, the solvent is hexafluoroisopropanol, and the concentration is 195 mg / mL;

[0130] The fourth spinning solution: The solute is a mixture of silk fibroin, PDO and osteoinductive material b, the content of PDO in the solute is 70 wt%, the content of osteoinductive material b in the solute is 4 wt%, the solvent is a mixture of hexafluoroisopropanol and formic acid with a volume ratio of 8:2, and the concentration is 205 mg / mL;

[0131] Osteoinductive material a, osteoinductive material b and osteoinductive material c: All are bioactive glasses;

[0132] (2) Using the electro-assisted solution blow spinning technology, the first micro-nano fiber membrane layer, the first transition layer, the second micro-nano fiber membrane layer, the second transition layer, the third micro-nano fiber membrane layer, the third transition layer, and the fourth micro-nano fiber membrane layer are successively formed on the receiving substrate;

[0133] When preparing the first micro-nano fiber membrane layer, the first spinning solution is pushed at a constant injection rate v 1 The process parameters include: the positive voltage is 16 kV, the negative voltage is 2 kV, v 1 is 7 mL / h, the air pressure is 7 atm, and the receiving distance is 17 cm; the thickness of the first micro-nano fiber membrane layer is 0.7 mm, the average pore size is 42 μm, the porosity is 83%, and the fiber alignment degree is 90%; the first micro-nano fiber membrane layer is used to guide the directional alignment of tendon fibroblasts and promote tendon differentiation and regeneration;

[0134] When preparing the first transition layer, the first spinning solution and the second spinning solution are simultaneously pushed. The injection rate of the first spinning solution decreases uniformly from v 1 to 0, and the injection rate of the second spinning solution increases uniformly from 0 to v 2 , and the sum of the injection rates of the first spinning solution and the second spinning solution at each moment remains unchanged; the process parameters include: the positive voltage is 16 kV, the negative voltage is 2 kV, v 2 is 7 mL / h, the injection time is 1 h, the air pressure is 7 atm, and the receiving distance is 17 cm;

[0135] When preparing the second micro-nano fiber membrane layer, the second spinning solution is pushed at a constant injection rate v 2 The process parameters include: the positive voltage is 16 kV, the negative voltage is 2 kV, the air pressure is 7 atm, and the receiving distance is 17 cm; the thickness of the second micro-nano fiber membrane layer is 0.7 mm, the average pore size is 73 μm, the porosity is 87%, and the fiber alignment degree is 50%; the second micro-nano fiber membrane layer is used to support and induce the differentiation and regeneration of unmineralized fibrous cartilage;

[0136] When preparing the second transition layer, the second spinning solution and the third spinning solution are simultaneously injected. The injection speed of the second spinning solution decreases uniformly from v 2 to 0, and the injection speed of the second spinning solution increases uniformly from 0 to v 3 . The sum of the injection speeds of the second spinning solution and the third spinning solution remains unchanged at each moment; the process parameters include: the positive voltage is 16 kV, the negative voltage is 2 kV, v 3 is 7 mL / h, the injection time is 1 h, the air pressure is 7 atm, and the receiving distance is 17 cm;

[0137] When preparing the third micro-nano fiber membrane layer, the third spinning solution is injected at a constant injection speed v 3 . The process parameters include: the positive voltage is 16 kV, the negative voltage is 2 kV, the air pressure is 7 atm, and the receiving distance is 17 cm; the thickness of the third micro-nano fiber membrane layer is 0.7 mm, the average pore diameter is 247 μm, the porosity is 90%, and the fiber alignment degree is 25%; the third micro-nano fiber membrane layer is used to induce mineralized fiber cartilage differentiation and regeneration;

[0138] When preparing the third transition layer, the third spinning solution and the fourth spinning solution are simultaneously injected. The injection speed of the third spinning solution decreases uniformly from v 3 to 0, and the injection speed of the second spinning solution increases uniformly from 0 to v 4 . The sum of the injection speeds of the third spinning solution and the fourth spinning solution remains unchanged at each moment; the process parameters include: the positive voltage is 16 kV, the negative voltage is 2 kV, v 4 is 7 mL / h, the injection time is 1 h, the air pressure is 7 atm, and the receiving distance is 17 cm;

[0139] When preparing the fourth micro-nano fiber membrane layer, the fourth spinning solution is injected at a constant injection speed v 4 . The process parameters include: the positive voltage is 16 kV, the negative voltage is 2 kV, the air pressure is 7 atm, and the receiving distance is 17 cm; the thickness of the fourth micro-nano fiber membrane layer is 0.7 mm, the average pore diameter is 420 μm, the porosity is 94%, and the fibers are completely randomly oriented; the fourth micro-nano fiber membrane layer is used to promote bone regeneration;

[0140] (3) A promoting osteogenic material c bulge is formed on the fourth micro-nano fiber membrane layer by using a heat treatment nanoimprint technology;

[0141] (3.1) Pour the photosensitive resin onto the nano-pattern master, spread it evenly with a roller until it completely covers the surface of the nano-pattern master, and then cure the photosensitive resin. After curing, a resin mold is formed on the nano-pattern master, and the nano-pattern master and the resin mold are separated;

[0142] (3.2) Deposit osteoinductive material c on the surface of the resin mold by electron beam evaporation technology;

[0143] (3.3) Perform plasma treatment on the resin mold with osteoinductive material c deposited on its surface, and simultaneously perform plasma treatment on the fourth micro-nano fiber membrane layer;

[0144] (3.4) Press the resin mold with osteoinductive material c deposited on its surface onto the fourth micro-nano fiber membrane layer. After the osteoinductive material c comes into contact with the fourth micro-nano fiber membrane layer, perform heat treatment. After the heat treatment is completed, remove the resin mold to obtain an integrated gradient bionic structure rotator cuff patch.

[0145] The total thickness of the finally obtained integrated gradient bionic structure rotator cuff patch is 4.9 mm; the protrusions of osteoinductive material c on the fourth micro-nano fiber membrane layer are cylindrical, with a diameter of 1500 nm and a height of 1200 nm. All the protrusions of osteoinductive material c are arranged in a dot matrix, and the distance between adjacent two protrusions of osteoinductive material c is 1500 nm; each transition layer and the two adjacent micro-nano fiber membrane layers form a continuous and unbroken structure, and the internal structure and composition change evenly without showing discrete stratification;

[0146] The peel strength of the integrated gradient bionic structure rotator cuff patch is 59.6 mN / mm; the energy dispersive spectroscopy analysis of each layer of the integrated gradient bionic structure rotator cuff patch is shown in Table 5:

[0147] Table 5

[0148]

[0149] As can be seen from Table 5, in the first micro-nano fiber membrane layer, silk fibroin provides N element as a nitrogen-containing component, while PDO dominates the proportion of C and O elements due to its high content; in the first transition layer, as the proportion of PDO further increases, the proportion of C element increases significantly, and at the same time the N content decreases due to the decrease in the proportion of silk fibroin; in the second micro-nano fiber membrane layer, since the solute only contains PDO, the proportion of C and O elements is close to the theoretical value of 3:2; in the second transition layer, due to the introduction of a small amount of bioactive glass (osteoinductive material a), signals of Si, Ca, and P elements begin to appear, but the C and O elements still mainly come from PDO; in the third micro-nano fiber membrane layer, as the proportion of bioactive glass (osteoinductive material a) increases, the signals of Si, Ca, and P elements increase significantly; in the third transition layer, the proportion of bioactive glass continues to increase, dominating the changes of Si, Ca, and P elements, and at the same time the presence of silk fibroin provides a small amount of N element; in the fourth micro-nano fiber membrane layer, the proportion of bioactive glass (osteoinductive material c) reaches the highest, the signals of Si, Ca, and P elements increase significantly, and the component of silk fibroin slightly increases the N element content.

[0150] Example 5

[0151] A preparation method of an integrated gradient bionic gradient structure rotator cuff patch, the specific steps are as follows:

[0152] (1)Preparation of materials;

[0153] The first spinning solution: The solute is a mixture of hyaluronic acid and PLCL, the content of PLCL in the solute is 90 wt%, the solvent is a mixture of hexafluoroisopropanol and acetic acid aqueous solution with a volume ratio of 7:3 (the volume fraction of acetic acid is 90%), and the concentration is 200 mg / mL;

[0154] The second spinning solution: The solute is PLCL, the solvent is hexafluoroisopropanol, and the concentration is 210 mg / mL;

[0155] The third spinning solution: The solute is a mixture of PLCL and osteoinductive material a, the content of osteoinductive material a in the solute is 2 wt%, the solvent is hexafluoroisopropanol, and the concentration is 220 mg / mL;

[0156] The fourth spinning solution: The solute is a mixture of hyaluronic acid, PLCL and osteoinductive material b, the content of PLCL in the solute is 90 wt%, the content of osteoinductive material b in the solute is 5 wt%, the solvent is a mixture of hexafluoroisopropanol and acetic acid aqueous solution with a volume ratio of 7:3 (the volume fraction of acetic acid is 90%), and the concentration is 230 mg / mL;

[0157] Osteoinductive material a, osteoinductive material b and osteoinductive material c: All are tricalcium phosphate;

[0158] (2)Using the electro-assisted solution blowing spinning technology to sequentially form the first micro-nano fiber membrane layer, the first transition layer, the second micro-nano fiber membrane layer, the second transition layer, the third micro-nano fiber membrane layer, the third transition layer, and the fourth micro-nano fiber membrane layer on the receiving substrate;

[0159] When preparing the first micro-nano fiber membrane layer, the first spinning solution is pushed at a constant injection speed v 1 for injection; The process parameters include: the positive voltage is 18 kV, the negative voltage is 3 kV, v 1 is 10 mL / h, the air pressure is 10 atm, and the receiving distance is 20 cm; The thickness of the first micro-nano fiber membrane layer is 1 mm, the average pore diameter is 50 μm, the porosity is 85%, and the fiber alignment degree is 88%; The first micro-nano fiber membrane layer is used to guide the directional arrangement of tendon fibroblasts and promote tendon differentiation and regeneration;

[0160] When preparing the first transition layer, the first spinning solution and the second spinning solution are pushed simultaneously. The injection speed of the first spinning solution decreases uniformly from v 1 to 0, and the injection speed of the second spinning solution increases uniformly from 0 to v 2, the sum of the injection rates of the first spinning solution and the second spinning solution at each moment remains unchanged; the process parameters include: a positive voltage of 18 kV, a negative voltage of 3 kV, v 2 is 10 mL / h, the injection time is 1 h, the air pressure is 10 atm, and the receiving distance is 20 cm;

[0161] When preparing the second micro-nano fiber membrane layer, the second spinning solution is injected at a constant injection rate v 2 ; the process parameters include: a positive voltage of 18 kV, a negative voltage of 3 kV, an air pressure of 10 atm, and a receiving distance of 20 cm; the thickness of the second micro-nano fiber membrane layer is 1 mm, the average pore size is 80 μm, the porosity is 90%, and the fiber alignment degree is 58%; the second micro-nano fiber membrane layer is used to support and induce the differentiation and regeneration of unmineralized fibrous cartilage;

[0162] When preparing the second transition layer, the second spinning solution and the third spinning solution are injected simultaneously. The injection rate of the second spinning solution decreases uniformly from v 2 to 0, and the injection rate of the third spinning solution increases uniformly from 0 to v 3 , and the sum of the injection rates of the second spinning solution and the third spinning solution at each moment remains unchanged; the process parameters include: a positive voltage of 18 kV, a negative voltage of 3 kV, v 3 is 10 mL / h, the injection time is 1 h, the air pressure is 10 atm, and the receiving distance is 20 cm;

[0163] When preparing the third micro-nano fiber membrane layer, the third spinning solution is injected at a constant injection rate v 3 ; the process parameters include: a positive voltage of 18 kV, a negative voltage of 3 kV, an air pressure of 10 atm, and a receiving distance of 20 cm; the thickness of the third micro-nano fiber membrane layer is 1 mm, the average pore size is 300 μm, the porosity is 92%, and the fiber alignment degree is 20%; the third micro-nano fiber membrane layer is used to induce the differentiation and regeneration of mineralized fibrous cartilage;

[0164] When preparing the third transition layer, the third spinning solution and the fourth spinning solution are injected simultaneously. The injection rate of the third spinning solution decreases uniformly from v 3 to 0, and the injection rate of the second spinning solution increases uniformly from 0 to v 4 , and the sum of the injection rates of the third spinning solution and the fourth spinning solution at each moment remains unchanged; the process parameters include: a positive voltage of 18 kV, a negative voltage of 3 kV, v 4 is 10 mL / h, the injection time is 1 h, the air pressure is 10 atm, and the receiving distance is 20 cm;

[0165] When preparing the fourth micro-nano fiber membrane layer, the fourth spinning solution is injected at a constant injection rate v4 Perform bolus injection; the process parameters include: positive voltage of 18 kV, negative voltage of 3 kV, air pressure of 10 atm, and receiving distance of 20 cm; the thickness of the fourth micro-nano fiber membrane layer is 1 mm, the average pore size is 500 μm, the porosity is 93%, and the fibers are completely randomly oriented; the fourth micro-nano fiber membrane layer is used to promote bone regeneration;

[0166] (3) Use the heat treatment nanoimprinting technology to form osteogenic material c protrusions on the fourth micro-nano fiber membrane layer;

[0167] (3.1) Pour the photosensitive resin onto the nano-patterned master, spread it evenly with a roller until it completely covers the surface of the nano-patterned master, then cure the photosensitive resin. After curing, a resin mold is formed on the nano-patterned master, and the nano-patterned master and the resin mold are separated;

[0168] (3.2) Use the electron beam evaporation technology to deposit osteogenic material c on the surface of the resin mold;

[0169] (3.3) Perform plasma treatment on the resin mold with osteogenic material c deposited on its surface, and at the same time perform plasma treatment on the fourth micro-nano fiber membrane layer;

[0170] (3.4) Press the resin mold with osteogenic material c deposited on its surface onto the fourth micro-nano fiber membrane layer. After the osteogenic material c contacts the fourth micro-nano fiber membrane layer, perform heat treatment. After the heat treatment is completed, remove the resin mold to obtain an integrated gradient bionic structure rotator cuff patch.

[0171] The total thickness of the finally obtained integrated gradient bionic structure rotator cuff patch is 6 mm; the osteogenic material c protrusions on the fourth micro-nano fiber membrane layer are cylindrical, with a diameter of 2500 nm and a height of 2500 nm. All the osteogenic material c protrusions are arranged in a dot matrix, and the distance between adjacent two osteogenic material c protrusions is 2000 nm; each transition layer and the two adjacent micro-nano fiber membrane layers form a continuous and unbroken structure, and the internal structure and composition change uniformly without showing discrete stratification;

[0172] The peel strength of the integrated gradient bionic structure rotator cuff patch is 55.8 mN / mm; the energy dispersive spectroscopy analysis of each layer of the integrated gradient bionic structure rotator cuff patch is shown in Table 6:

[0173] Table 6

[0174]

[0175] As can be seen from Table 6, in the first micro-nano fiber membrane layer, PLCL dominates the proportion of C and O elements due to its relatively high content, while hyaluronic acid provides a small amount of N element; in the first transition layer, as the proportion of PLCL increases, the proportion of C element rises, and at the same time, the N content decreases due to the decrease in the proportion of hyaluronic acid; in the second micro-nano fiber membrane layer, since the solute only contains PLCL, the proportion of C and O elements is close to the theoretical value of 3:2; in the second transition layer, due to the introduction of a small amount of tricalcium phosphate (osteogenic material a), Ca and P element signals begin to appear, but the C and O elements still mainly come from PLCL; in the third micro-nano fiber membrane layer, as the content of tricalcium phosphate (osteogenic material a) increases, the Ca and P signals are significantly enhanced, and the proportion of C element decreases accordingly; in the third transition layer, the proportion of tricalcium phosphate further increases, resulting in a significant increase in the Ca and P content, and at the same time, hyaluronic acid contributes a small amount of N element; in the fourth micro-nano fiber membrane layer, the proportion of tricalcium phosphate (osteogenic material c) reaches the highest, the Ca and P signal intensity is the largest, and the proportion of C element is further reduced.

[0176] Comparative Example 1

[0177] A preparation method of a patch, which is only different from that of Example 1 in that: in step (2), the first transition layer, the second transition layer, and the third transition layer are not formed on the receiving substrate, and the first micro-nano fiber membrane layer, the second micro-nano fiber membrane layer, the third micro-nano fiber membrane layer, and the fourth micro-nano fiber membrane layer are directly formed on the receiving substrate. When preparing the first to fourth micro-nano fiber membrane layers, the conditions are set the same as those in Example 1.

[0178] The peel strength of the finally prepared patch is 15.6 mN / mm.

[0179] Comparative Example 2

[0180] A preparation method of a patch, which is only different from that of Example 2 in that: in step (2), the first transition layer, the second transition layer, and the third transition layer are not formed on the receiving substrate, and the first micro-nano fiber membrane layer, the second micro-nano fiber membrane layer, the third micro-nano fiber membrane layer, and the fourth micro-nano fiber membrane layer are directly formed on the receiving substrate. When preparing the first to fourth micro-nano fiber membrane layers, the conditions are set the same as those in Example 2.

[0181] The peel strength of the finally prepared patch is 16.9 mN / mm.

[0182] Comparative Example 3

[0183] A preparation method of a patch, which is only different from that of Example 3 in that: in step (2), the first transition layer, the second transition layer, and the third transition layer are not formed on the receiving substrate, and the first micro-nano fiber membrane layer, the second micro-nano fiber membrane layer, the third micro-nano fiber membrane layer, and the fourth micro-nano fiber membrane layer are directly formed on the receiving substrate. When preparing the first to fourth micro-nano fiber membrane layers, the conditions are set the same as those in Example 3.

[0184] The peel strength of the finally obtained patch is 21.8 mN / mm.

[0185] Comparative Example 4

[0186] A method for preparing a patch, which is only different from Example 4 in that: in step (2), the first transition layer, the second transition layer, and the third transition layer are not formed on the receiving substrate, and the first micro-nano fiber film layer, the second micro-nano fiber film layer, the third micro-nano fiber film layer, and the fourth micro-nano fiber film layer are directly formed on the receiving substrate. When preparing the first to fourth micro-nano fiber film layers, the conditions are set the same as in Example 4.

[0187] The peel strength of the finally obtained patch is 20.9 mN / mm.

[0188] Comparative Example 5

[0189] A method for preparing a patch, which is only different from Example 5 in that: in step (2), the first transition layer, the second transition layer, and the third transition layer are not formed on the receiving substrate, and the first micro-nano fiber film layer, the second micro-nano fiber film layer, the third micro-nano fiber film layer, and the fourth micro-nano fiber film layer are directly formed on the receiving substrate. When preparing the first to fourth micro-nano fiber film layers, the conditions are set the same as in Example 5.

[0190] The peel strength of the finally obtained patch is 18.9 mN / mm.

[0191] Compared with Comparative Examples 1-5 and Examples 1-5, the peel strength of the patch decreased significantly. This indicates that the presence of the transition layer plays a key role in enhancing the interfacial bonding force of the patch. By achieving a uniform transition of the material composition and structure of each layer, it effectively avoids the discreteness between layers and significantly improves the overall mechanical properties of the patch. In contrast, the method of directly preparing adjacent micro-nano fiber layers in the comparative examples lacks the component transition link, resulting in a weak interfacial bonding force between layers. Under external forces, interlayer separation is more likely to occur, causing a significant reduction in the peel strength of the patch, which is not conducive to the patch playing a lasting and stable repair effect in practical applications.

Claims

1. A method for preparing an integrated gradual bionic gradient structure rotator cuff patch, characterized in that: After sequentially forming a first micro-nano fiber membrane layer, a first transition layer, a second micro-nano fiber membrane layer, a second transition layer, a third micro-nano fiber membrane layer, a third transition layer, and a fourth micro-nano fiber membrane layer on a receiving substrate by using an electrically assisted solution blowing spinning technique, a bone promoting material protrusion is formed on the fourth micro-nano fiber membrane layer by using a heat treatment nanoimprinting technique; When preparing the i-th micro-nano fiber membrane layer, the corresponding i-th spinning solution is injected at a constant injection speed v i Perform bolus injection, i=1,2,3; When preparing the i+1th micro-nano fiber membrane layer, the corresponding i+1th spinning solution is injected at a constant injection speed v i+1 Give a bolus injection; The process of preparing the i-th transition layer is as follows: the i-th spinning solution and the i+1-th spinning solution are injected simultaneously, and the injection speed of the i-th spinning solution is determined by v i The speed of the spinning solution is uniformly reduced to 0, and the injection speed of the i+1 spinning solution is uniformly increased from 0 to v i+1 , the sum of the injection speed of the i-th spinning solution and the injection speed of the i+1-th spinning solution at each moment remains unchanged; The first micro-nano fiber membrane layer is used to guide the directional arrangement of tendon fibroblasts to promote tendon differentiation and regeneration; the second micro-nano fiber membrane layer is used to support and induce the differentiation and regeneration of unmineralized fibrocartilage; the third micro-nano fiber membrane layer is used to induce the differentiation and regeneration of mineralized fibrocartilage; the fourth micro-nano fiber membrane layer is used to promote bone regeneration; the osteogenic material protrusions are cylindrical, with a diameter of 100-2500nm and a height of 5-2500nm. All osteogenic material protrusions are arranged in a lattice shape, and the distance between two adjacent osteogenic material protrusions is 100-2000nm; Each transition layer and the two adjacent micro-nano fiber membrane layers form a continuous and unbroken structure, and the internal structure and composition change evenly and transitionally without showing discrete stratification.

2. The method for preparing a one-piece gradual bionic gradient structure rotator cuff patch according to claim 1, characterized in that: The average pore size of the first micro-nano fiber membrane layer is 10-50 μm, the porosity is 70-85%, the orientation degree of the fiber arrangement is >80%, the fiber composition is a mixture of natural polymer materials and synthetic polymer materials, and the content of the synthetic polymer materials is 10-90wt%.

3. The method for preparing a one-piece gradual bionic gradient structure rotator cuff patch according to claim 1, characterized in that: The average pore size of the second micro-nano fiber membrane layer is 30-80 μm, the porosity is 80-90%, the orientation degree of the fiber arrangement is 50-60%, and the fiber component is a synthetic polymer material.

4. The method for preparing a one-piece gradual bionic gradient structure rotator cuff patch according to claim 1, characterized in that: The average pore size of the third micro-nano fiber membrane layer is 100-300 μm, the porosity is 85-95%, the orientation degree of the fiber arrangement is greater than 20% and less than 30%, the fiber component is a mixture of synthetic polymer materials and osteogenic materials, and the content of osteogenic materials is 1-2wt%.

5. The method for preparing a one-piece gradual bionic gradient structure rotator cuff patch according to claim 1, characterized in that: The average pore size of the fourth micro-nano fiber membrane layer is 200-500μm, the porosity is >90%, the fibers are arranged in a completely random orientation, and the fiber composition is a mixture of natural polymer materials, synthetic polymer materials and osteogenic materials. The content of synthetic polymer materials is 10-90wt%, and the content of osteogenic materials is 2-5wt%.

6. The method for preparing a one-piece gradual bionic gradient structure rotator cuff patch according to claim 1, characterized in that: The thickness of each micro-nano fiber membrane layer ranges from 0.1 to 1 mm, and the total thickness of the integrated gradual bionic gradient structure rotator cuff patch is 0.7 to 6 mm.

7. A method for preparing an integrated gradual bionic gradient structure rotator cuff patch according to any one of claims 1 to 6, characterized in that: The specific steps of forming the protrusions of the osteogenic material on the fourth micro-nano fiber membrane layer by using the heat treatment nanoimprinting technology are as follows: (a) pouring a photosensitive resin onto a nano-pattern master, spreading it evenly with a roller until it completely covers the surface of the nano-pattern master, curing the photosensitive resin, forming a resin mold on the nano-pattern master after the curing, and separating the nano-pattern master from the resin mold; (b) Depositing osteogenic material on the surface of the resin mold using electron beam evaporation technology; (c) plasma treating the resin mold with the osteogenic material deposited on the surface, and simultaneously plasma treating the fourth micro-nanofiber membrane layer; (d) Pressing the resin mold with the osteogenic material deposited on the surface onto the fourth micro-nano fiber membrane layer so that the osteogenic material contacts the fourth micro-nano fiber membrane layer, performing heat treatment, and removing the resin mold after the heat treatment is completed.

8. The method for preparing a one-piece gradual bionic gradient structure rotator cuff patch according to claim 7, characterized in that: The process parameters of electrically assisted solution blowing spinning include: positive voltage of 10-18 kV, negative voltage of 1-3 kV, injection speed of 1-10 mL / h, air pressure of 1-10 atm, and receiving distance of 10-20 cm.

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